Bacterial infections constitute one of the major health problems worldwide. A gradual increase in the resistance to antibiotics leads to a serious thread for successful treatment of bacterial infections. This feature has stimulated the interest in vaccines to prevent infections. Polysaccharides, forming a thick capsule that surrounds the bacterial pathogen, represent a major determinant of pathogenicity. Therefore, development of polysaccharide-based vaccines provides an attractive approach for fighting the infectious diseases. In contrast to pure polysaccharides, carbohydrates coupled to an immunogenic protein present enhanced immunogenicity. Glycoconjugate vaccine provides a long lasting protection for adults as well as for persons at high risk and young children. The traditional chemical producing approach has enabled the production of several highly successful conjugated vaccines in clinical use. However, the traditional approach suffers from multiple fermentations, purification steps, low yields and non-specific chemical conjugation, thus leading to high costs for vaccine production. The objective of this application is to explore two recently established bacterial protein glycosylation systems to obtain glycoconjugate vaccines in a facile, efficient, and easily applicable manner. These bacterial glycosylation systems comprise two highly promiscuous proteins (PglB for N-glycosylation and PglL for O-glycosylation), which catalyze the transfer of polysaccharides from a diphospho-lipid donor to target proteins. The proof-of-concept experiments conducted by our lab and other groups lend compelling evidence for further exploration of such a system in the glyconjugate vaccine development. Furthermore, the success of this proposed research also heavily relies on the in-depth understanding of polysaccharide biosynthesis in bacteria, which we have been studying in the past several years with a combination of genetic, chemical, and biochemical approaches. To fulfill this objective, we proposed three specific aims: Aim 1. Investigation of PglB catalyzed N-glycosylation system The glycoconjugate synthesis of E. coli O157:H7 O-antigen attached to a commonly used carrier protein (CRM197) by PglB involved in N-glycosylation system will be demonstrate in vitro and in vivo. Based on the chemical synthesis of lipid pyrophosphate linked monosaccharides, enzymatic assembly of the corresponding O-antigen repeating unit, reconstitution of O-antigen polymerization, and over-expression and purification of PglB and CRM197, an in vitro reaction system will be established. With this system, CRM197 will be engineered to be a PglB acceptor substrate and certain scale of glycoconjugates will be obtained for characterization. Then development of an in vivo fermentation system that contains all the necessary components for glycoconjugate production will be explored. E. coli K12 W3110 will be utilized as a host strain. Gene waaL responsible for ligation of nascent polysaccharide chains to the core-Lipid A will be disrupted to accumulate O-antigen chains in the periplasm. Furthermore, E. coli O157 LPS biosynthesis gene cluster will be introduced into E. coli K12 W3110 waaL strain. Finally, CRM197 variants obtained from in vitro study will be co-expressed with PglB in this engineered E. coli model strain to produce O- antigen-CRM197 glycoconjugate. Regulation of O-antigen chain length will also be explored. Aim 2. Investigation of PglL catalyzed O-glycosylation system PglB only tolerates glycan donor with a C2 acetamido group at the reducing end sugar while PglL was showed to be able to transfer glycan donor with hexose at the reducing end. Since the majority of CPS glycans contain hexose at the reducing end, PglL has higher potential for the generation of CPS conjugate vaccines than PglB. We choose S. pneumoniae type 14 as a model to demonstrate PglL mediated glycoconjugate vaccine production. The repeating unit of CPS from this strain (CPS14) will be reconstructed in vitro and used as sugar donor for in vitro reaction. The importance of lipid moiety for PglL recognition will be investigated. The substrate requirement and peptide preference for PglL protein acceptor will be determined and the commonly used carrier protein CRM197 will be engineered into a PglL substrate with the knowledge obtained from these studies. Then the feasibility of heterologously expressing of CPS14 in the host strain will then be investigated. Genes encoding PglL and the favorable CRM197 variants, along with the CPS14 gene cluster required for the heterologous expression of CPS14, will be introduced into the mutant host strain. Production and purification of CPS14-CRM197 conjugates from fermentation of this engineered strain will be explored. Aim 3. Quality control and bioactivity assay of polysaccharide conjugated vaccines A series of techniques including NMR, MS, CD will be employed to characterize the glycoconjugate vaccines produced in our systems. Bioactivity of the vaccines will be further evaluated in mice models. Successful demonstration of the production of CRM197 conjugated with E. coli O157:H7 O-antigen and S. pneumoniae type14 CPS will set the groundwork for producing glyconjugate vaccine for other pathogens. The accessibility of polysaccharide biosynthesis loci will enable generalization of our approach for the production of a variety of polysaccharide conjugate vaccines. PUBLIC HEALTH RELEVANCE: Bacterial infections are one of the major health problems worldwide. Polysaccharides, forming a thick capsule that surrounds the bacterial pathogen, represent a major determinant of pathogenicity. With the increasing emergence of resistance toward major antibiotics, development of polysaccharide-based vaccines provides an attractive approach for fighting the infectious diseases. Polysaccharides are better to be conjugated to a carrier protein as conjugate vaccines to enhance their efficacy. The traditional chemical approach of polysaccharide conjugate vaccine production has enabled the production of several highly successful conjugated vaccines currently in clinical use. However, the traditional method suffers from complex production steps, low yields and impure products, thus leading to high costs for vaccine production. The objective of this application is to develop a novel method for polysaccharide conjugate vaccine production. With this method, we can obtain polysaccharide conjugate vaccines in a facile, efficient, and easily applicable manner. This method will firstly be explored with model pathogens. Then the established method can be easily applied to other pathogens.